WO2022124418A1 - Anticorps capable de se lier spécifiquement à un domaine actif d'exonucléase 5'→ 3' de l'adn polymérase - Google Patents

Anticorps capable de se lier spécifiquement à un domaine actif d'exonucléase 5'→ 3' de l'adn polymérase Download PDF

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WO2022124418A1
WO2022124418A1 PCT/JP2021/045700 JP2021045700W WO2022124418A1 WO 2022124418 A1 WO2022124418 A1 WO 2022124418A1 JP 2021045700 W JP2021045700 W JP 2021045700W WO 2022124418 A1 WO2022124418 A1 WO 2022124418A1
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amino acid
antibody
acid sequence
fragment
seq
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PCT/JP2021/045700
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English (en)
Japanese (ja)
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峻史 吉兼
信幸 黒澤
正治 磯部
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東洋紡株式会社
国立大学法人富山大学
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Priority to JP2022568364A priority Critical patent/JPWO2022124418A1/ja
Priority to CN202180082185.3A priority patent/CN116583600A/zh
Priority to EP21903514.4A priority patent/EP4261226A1/fr
Priority to US17/996,601 priority patent/US11970549B2/en
Publication of WO2022124418A1 publication Critical patent/WO2022124418A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Nucleic acid amplification methods especially antibodies that specifically bind to the 5' ⁇ 3'exonuclease active domain of DNA polymerase used in polymerase chain reaction (hereinafter referred to as "PCR") and related technologies. Provided.
  • PCR polymerase chain reaction
  • the synthesis of DNA from a nucleic acid template using DNA polymerase is used and applied to various methods such as sequencing method and nucleic acid amplification method in the field of molecular biology.
  • sequencing method and nucleic acid amplification method in the field of molecular biology.
  • nucleic acid amplification method has already been put into practical use not only in the research field but also in the forensic field such as genetic diagnosis and paternity testing, or in the microbiological examination in foods and the environment.
  • a typical nucleic acid amplification method is PCR.
  • PCR is performed by (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) the primer using a DNA polymerase.
  • This is a method in which the target nucleic acid in the sample is amplified by repeating the three steps of elongation as one cycle. In some cases, (2) annealing and (3) elongation are performed at the same temperature in one step, and two steps are regarded as one cycle.
  • PCR is widely used in medical / biological research and clinical diagnosis because of its characteristics such as sensitivity that can be amplified from a nucleic acid sample equivalent to one copy in principle and several copies in reality, and specificity that amplifies only a specific part. I came.
  • PCR is being further developed, such as the Multiplex PCR method that simultaneously amplifies multiple primers, and the real-time PCR method that monitors the production process of amplification products over time using fluorescent dyes and fluorescently labeled probes.
  • the Multiplex PCR method that simultaneously amplifies multiple primers
  • the real-time PCR method that monitors the production process of amplification products over time using fluorescent dyes and fluorescently labeled probes.
  • nucleic acid amplification methods are widely used for gene analysis of large samples such as HTS (High Throughput Screening), food inspections and environmental inspections that require processing of multiple samples.
  • HTS High Throughput Screening
  • the nucleic acid amplification reaction solution is left for a long time (for example, several hours to several days) after preparation.
  • the reaction solution is left at room temperature, there is a concern that the stability of the reaction solution may decrease.
  • the Ct (Threshold cycle) value is delayed or the Ct value detection itself is delayed by leaving the prepared reaction solution at room temperature. A plurality of cases of the impossible phenomenon have been confirmed (Patent Documents 1 and 2).
  • the present inventors have previously found a problem that nucleic acid templates, primers, probes and the like used in nucleic acid amplification methods and the like are degraded in the coexistence with a DNA polymerase having a 5' ⁇ 3'exonuclease active domain. There is.
  • the main object of the present invention is to provide an antibody (specifically binding) to the 5' ⁇ 3'exonuclease active domain of DNA polymerase or a fragment thereof, and a method for producing the antibody or fragment thereof.
  • the present inventors have conducted an antibody or fragment thereof (specifically binding) to the 5' ⁇ 3'exonuclease active domain of DNA polymerase, and the antibody or fragment thereof. We have found a useful manufacturing method for. The present invention has been completed as a result of further diligent research based on these findings.
  • Typical inventions are as follows.
  • X B3 is F, Y, L, I, G, S, T, D, or N.
  • X B4 is G, S, T, D, N, K, R, or H.
  • X B5 is G, S, T, or A,
  • X B6 is G, S, T, D, or N,
  • X B7 is S, T, F, Y, D, N, K, R, or H.
  • X B8 is S, T, V, L, I, or M] Heavy chain CDR2 consisting of the amino acid sequence represented by The following formulas (C-1) to (C-6): VRX C1 X C2 X C3 GX C4 X C5 X C6 TGFDX C7 (C-1) VRX C1 X C2 X C3 GX C4 X C5 X C6 FDX C7 (C-2) X C8 RDGALGLAVNWFDX C7 (C-3) ATSDDYYALNI (C-4) TTAYYSRYSYMYMFDX C7 (C-5) TTALRDX C7 (C-6) [During the ceremony, X C1 is A, S, D, K, H, or R.
  • X C2 is G, A, D, P, K, H, or R.
  • X C3 is S, T, L, Y, or I
  • X C4 is A, V, I, R, or L
  • X C5 is A, V, Y, or P
  • X C6 is A, V, S, T, or Y
  • X C7 is V, I, L, S, T, or N
  • X C8 is A or V]
  • X D6 is F, Y, or W]
  • Light chain CDR1 consisting of the amino acid sequence represented by The following formula (E-1): X E1 X E2 X E3 (E-1) [During the ceremony, X E1 is G, S, T, D, N, F, Y, K, R, or H.
  • X E2 is G, A, S, T, V, L or I
  • X E3 is K, R, H, D, N, S, or T]
  • Light chain CDR2 consisting of the amino acid sequence represented by The following formula (F-1) or (F-2): X F1 X F2 X F3 X F4 X F5 X F6 X F7 X F8 (F-1) X F1 X F2 X F3 X F4 X F5 X F6 X F7 X F8 X F9 (F-2) [During the ceremony, X F1 is L, I, E, Q, F, Y or W.
  • X F2 is D, N, E or Q
  • X F3 is S, T, F, or Y
  • X F4 is G, S, T, F, Y, N or Q
  • X F5 is S, T, N, Q, L or I
  • X F6 is G, S, T, F, Y, or W
  • X F7 is S, T, P, Y or W
  • X F8 is L, I, P, K, R, H, Y, T, or W
  • X F9 is G, S, T, D or E]
  • X B41 is G, S, T, D, or H
  • X B51 is G or S and is X B61 is G, S, T, or D
  • X B71 is S, T, Y, D, or H
  • X B81 is S, T, V, I, or M]
  • Heavy chain CDR2 consisting of the amino acid sequence represented by The following formulas (C-1-1) to (C-6-1): VRX C11 X C21 X C31 GX C41 X C51 X C61 TGFDX C71 (C-1-1) VRX C11 X C21 X C31 GX C41 X C51 X C61 FDX C71 (C-2-1)
  • X C81 RDGALGLAVNWFDX C71 (C-3-1) ATSDDYYALNI (C-4) TTAYYSRYSYMYMFDX C71 (C-5-1) TTALRDX C71 (C-6-1) [During the ceremony, X C11 is A or R,
  • X E21 is A, T, or V
  • X E31 is K, D, N, or S
  • Light chain CDR2 consisting of the amino acid sequence represented by The following formula (F-1-1) or (F-2-1): X F11 QX F31 X F41 X F51 X F61 X F71 X F81 (F-1-1) X F11 QX F31 X F41 X F51 X F61 X F71 X F81 T (F-2-1)
  • XF11 is L, Q, F, or Y
  • X F31 is S or Y
  • X F41 is G, N, Q, or Y
  • X F51 is S, N, or I
  • X F61 is G, S, Y, or W.
  • X F71 is S, P, or W
  • X F81 is L, P, H, Y, or T
  • the antibody or fragment thereof according to Item 5 which comprises a sequence region adjacent to the C-terminal of the light chain CDR2, which comprises the amino acid sequence represented by.
  • Item 7 Item 6.
  • Item 8 Item 6.
  • Amino acid region B Amino acid region C selected from the 161 to 182nd region from the N-terminal of SEQ ID NO: 1 or the 162 to 183th region from the N-terminal of SEQ ID NO: 2 or 3; From the N-terminal of SEQ ID NO: 1.
  • An antibody or fragment thereof present in any region of amino acid region D selected from the region of positions 269 to 285 or the region of positions 271 to 287 of SEQ ID NO: 2 or 3.
  • Item 11 Item 6. The antibody or fragment thereof according to Item 10, wherein the at least one epitope is present in any of the amino acid regions A and B.
  • the epitope in the amino acid region A is any of SEQ ID NOs: 60 to 63
  • the epitope in the amino acid region B is SEQ ID NO: 64 or 65
  • the epitope in the amino acid region C is any of SEQ ID NOs: 66 to 74.
  • Item 10 The antibody or fragment thereof according to Item 10 or 11, wherein the epitope in the amino acid region D is any of SEQ ID NOs: 75 to 83.
  • the epitope in the amino acid region A is SEQ ID NO: 61 or 62
  • the epitope in the amino acid region B is SEQ ID NO: 64 or 65
  • the epitope in the amino acid region C is SEQ ID NO: 66, 67, 68, 70, or 71.
  • Item 6 The antibody or fragment thereof according to any one of Items 10 to 12, wherein the epitope in the amino acid region D is SEQ ID NO: 77, 78, 80, or 82.
  • Item 14 Item 6.
  • the heavy chain CDR3 consists of the amino acid sequence shown in any of SEQ ID NOs: 21 to 28 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • Item 6 The item according to any one of Items 1 to 14, wherein the light chain CDR3 consists of an amino acid sequence shown in any of SEQ ID NOs: 42 to 48 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences. An antibody or fragment thereof.
  • the heavy chain CDR3 comprises the amino acid sequence set forth in any of SEQ ID NOs: 21-28. Item 6.
  • a heavy chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 4 to 11 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • a heavy chain CDR2 consisting of the amino acid sequence shown in any of SEQ ID NOs: 12 to 20 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • a heavy chain CDR3 consisting of the amino acid sequence shown in any of SEQ ID NOs: 21 to 28 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • a light chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 29 to 35 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • Light chain CDR2 consisting of the amino acid sequence represented by any of YTN, YTD, YAD, YAN, DAS, GVK, RAK, GAK, or TAS, and Item 6.
  • Heavy chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 4 to 11 and Heavy chain CDR2 consisting of the amino acid sequence shown in any of SEQ ID NOs: 12 to 20 and Heavy chain CDR3 consisting of the amino acid sequence shown in any of SEQ ID NOs: 21 to 28, Light chain CDR1 consisting of the amino acid sequence shown in any of SEQ ID NOs: 29 to 35, Light chain CDR2 consisting of the amino acid sequence represented by any of YTN, YTD, YAD, YAN, DAS, GVK, RAK, GAK, or TAS, and Item 6.
  • it comprises a sequence region adjacent to the C-terminal of the light chain CDR2 consisting of the amino acid sequence shown in any of SEQ ID NOs: 36 to 41 or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences.
  • Item 21 Item 6. The antibody or fragment thereof according to any one of Items 15, 17, and 19, wherein the mutation is a conservative substitution.
  • Item 22 Item 2. A fragment of the antibody according to any one of Items 1 to 21, which is Fab, F (ab') 2, or scFv (antigen-binding fragment).
  • Item 23 Item 2. A reagent containing the antibody according to any one of Items 1 to 21 or a fragment thereof, or the fragment according to Item 22.
  • Item 24 Item 23.
  • Item 25 Item 6.
  • Item 26 Item 6.
  • [Item 27] A method for producing an antibody or a fragment thereof (antibody-binding fragment) that specifically binds to the 5' ⁇ 3'exonuclease active domain of DNA polymerase, and is a part of DNA polymerase (here, the part is 5' ⁇ A production method comprising step A of selecting an antibody having the ability to bind to the whole DNA polymerase from an antibody produced by an animal immunized with an immunogen (including an exonuclease active domain).
  • the immunogen comprises a 5' ⁇ 3'exonuclease active domain of DNA polymerase.
  • Step A is the ability of an antibody produced by an immunogen-immunized animal consisting of a portion of the Tth polymerase (where the portion comprises a 5' ⁇ 3'exonuclease active domain) to bind to the entire Taq polymerase.
  • Item 27 The production method according to Item 27, which is a step of selecting an antibody having.
  • Step A is a step of selecting an antibody having a binding ability to the whole Taq polymerase from an antibody produced by an immunogen immunized with an immunogen consisting of a 5' ⁇ 3'exonuclease active domain of Tth polymerase. The manufacturing method described.
  • Step A Production of an antibody fragment (antigen-binding fragment) comprising a step of expressing a part of the amino acid sequence by a genetic engineering technique based on the amino acid sequence of the antibody obtained by the production method according to any one of Items 27 to 32. Method.
  • Method Item 34] Item 2.
  • Substrate DNA at 25 ° C. for 24 hours where the substrate DNA may be single-stranded or double-stranded, and the substrate DNA may have a function as a probe
  • 5' ⁇ Item 6 The antibody or fragment thereof according to any one of Items 1 to 21 and 34, or Item 22 in which the substrate DNA degradation rate is 40% or less when coexisting with a DNA polymerase having a 3'exonuclease active domain.
  • a reagent for stabilizing a composition comprising a DNA polymerase having a 5' ⁇ 3'exonuclease active domain and at least one nucleic acid selected from the group consisting of primers, probes, and nucleic acid templates.
  • a method for stabilizing a composition comprising a DNA polymerase having a 5' ⁇ 3'exonuclease active domain and at least one nucleic acid selected from the group consisting of primers, probes, and nucleic acid templates, wherein the composition comprises 1 to 1 to 3.
  • a method comprising the step of adding the antibody or fragment thereof according to any one of 21, 34, and 35, or the fragment according to Item 22, to the composition.
  • the present invention provides an antibody or fragment thereof that specifically binds to the 5' ⁇ 3'exonuclease active domain of DNA polymerase, and a useful method for producing the antibody or fragment thereof.
  • a useful method for producing the antibody or fragment thereof for example, by adding the antibody or a fragment thereof to a reagent containing a DNA polymerase having a 5' ⁇ 3'exonuclease active domain and nucleic acids such as primers and probes, the degradation of the nucleic acid can be suppressed and the stability of the reagent can be suppressed. Can be improved.
  • the target nucleic acid when amplified by using the reagent, the non-specific amplification of the target nucleic acid can be suppressed and the target nucleic acid can be amplified with high efficiency by suppressing the generation of fragments due to the decomposition of the nucleic acid. Even a very small amount of target nucleic acid can be detected with high sensitivity.
  • the amino acid may be a natural amino acid or an unnatural amino acid.
  • unnatural amino acids include citrulline, ornithine, ⁇ -acetyl-lysine, ⁇ -alanine, aminobenzoic acid, 6-aminocaproic acid, aminobutyric acid, hydroxyproline, mercaptopropionic acid, 3-nitrotyrosine, norleucine, pyroglutamic acid and the like.
  • the amino acid may be, for example, L-amino acid, D-amino acid, or DL-amino acid.
  • the amino acid sequence identity refers to the degree of amino acid matching when two or more amino acid sequences to be compared are optimally aligned.
  • the identity of the amino acid sequence can be calculated using a commercially available or telecommunications line (Internet) available analysis tool, eg, using the commercially available software GENETYX (Genetics, Inc.), or National Biotechnology Information Systems. Calculate using the default (initial setting) parameters in the BLAST (Basic local alignment software) (http://www.ncbi.nlm.nih.gov/BLAST/) homology algorithm of the National Center for Biotechnology Information (NCBI). Can be done.
  • BLAST Basic local alignment software
  • amino acid sequences disclosed herein lack one or more (eg, 1, 2, or 3) amino acids in the sequence unless they inhibit the binding of the DNA polymerase to the 5' ⁇ 3'exonuclease domain. It may be lost, substituted, or modified, and one or more (eg, 1, 2, or 3) amino acids may be inserted or added to the sequence.
  • amino acid substitution is preferably a substitution (conservative substitution) with another amino acid having a similar structure and / or property.
  • Conservative substitutions include, for example, substitutions within the group shown in Table 1.
  • amino acid modifications include modification of functional groups such as amino groups, carboxyl groups, hydroxyl groups, and sulfhydryl (SH) groups.
  • Modifications of the functional groups include, for example, glycosylation; methylation; esterification; amidation; PEGylation; phosphorylation; hydroxylation; t-butoxycarbonyl (Boc) group, 9-fluorenylmethyloxycarbonyl (Fmoc) group.
  • Binding to protective groups such as; biotinylation; binding to fluorescent dyes such as fluorescein isothiocyanate (FITC); binding to enzymes such as peroxidase (HRP), alkali phosphatase (ALP) and the like.
  • the antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
  • the antibody can be any isotype, such as IgG, IgA, IgD, IgE, IgM and the like. Examples of the antibody include, but are not limited to, mouse antibody, rat antibody, guinea pig antibody, human antibody and the like.
  • the antibody may be a chimeric antibody such as a guinea pig-mouse chimeric antibody or a mouse-human chimeric antibody.
  • the antibody fragment is not particularly limited as long as it contains heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3, and is not particularly limited, for example, Fv, Fab, Fab', (Fab') 2 , scFv, scFv-Fc. , Diabody, triabody, tetrabody, minibody and the like.
  • the antibody fragment is a fragment having an antigen-binding ability (antigen-binding fragment).
  • the heavy chain CDR1 to 3 and the light chain CDR1 to 3 are specified by a homology search by IMGT / BlastSearch (http://www.imgt.org/blast/).
  • Nucleotides such as DNA and RNA may be analogs to which known chemical modifications are applied as exemplified below.
  • a hydrolyzing enzyme such as a nuclease
  • the phosphate residue (phosphate) of each nucleotide is replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate phosphorodithionate
  • the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is designated as -OR (R is, for example, -CH 3 , -CH 2 CH 2 OCH 3 , -CH 2 CH 2 NHC (NH) NH 2 , -CH. 2 CONHCH 3 , -CH 2 CH 2 CN, etc.) may be substituted.
  • the base moiety pyrimidine, purine
  • examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto.
  • domain E An antibody that specifically binds to the 5' ⁇ 3'exonuclease active domain of DNA polymerase (hereinafter referred to as "domain E")
  • domain E The antibody of the present invention or a fragment thereof shall specifically bind to domain E of DNA polymerase. Therefore, 5' ⁇ 3'exonuclease activity can be inhibited, and it is useful as a 5' ⁇ 3'exonuclease activity inhibitor. Since the 5' ⁇ 3'exonuclease activity inhibitor of the present invention is an antibody, it has a high specificity and can be inactivated by heating or the like, so that it has an advantage that it can be easily applied to a hot start method. ..
  • the DNA polymerase is not particularly limited as long as it has domain E.
  • the DNA polymerase may be a wild-type DNA polymerase, or may be a recombinant DNA polymerase obtained by introducing a gene encoding the DNA polymerase into an arbitrary host cell, or a DNA obtained by modifying the gene. It may be a polymerase.
  • the DNA polymerase may be a DNA polymerase in which domain E is fused with a DNA polymerase that does not have domain E in the wild type.
  • the DNA polymerase is preferably a thermostable DNA polymerase.
  • heat resistance refers to the property of retaining preferably 50% or more of DNA polymerase activity even when heat-treated at a high temperature such as at 60 ° C. for 30 minutes.
  • the heat-resistant DNA polymerase include DNA polymerase derived from Thermus aquaticus (Taq polymerase), DNA polymerase derived from Thermus thermophilus HB8 (Tth polymerase), DNA polymerase derived from Thermus sp Z05 (Z05 polymerase), and DNA derived from Bacillus caldotenax.
  • Bca polymerase DNA polymerase derived from Bacillus theatermophilus (Bst polymerase), DNA polymerase derived from Thermococcus kodakarensis (KOD polymerase), DNA polymerase derived from Pyrococcus furiosus (Pfu polymerase), DNA polymerase derived from Pyrococos DNA polymerase derived from blockianus (Tbr polymerase), DNA polymerase derived from Thermus filiformis (Tfi polymerase), DNA polymerase derived from Thermus flavus (Tfl polymerase), DNA polymerase derived from Thermotoga maritima (Tma polymerase), Thermotoga Tne polymerase), DNA polymerase derived from Thermococcus litoralis (Vent polymerase), DNA polymerase derived from Pyrococos GB-D (DEEPVENT polymerase), and the like, but the present invention is not limited thereto.
  • the term such as Taq polymerase also includes a mutant.
  • the variant consists of an amino acid sequence having 80% or more identity with the amino acid sequence of the original DNA polymerase, and enzymatics such as polymerase activity, 5' ⁇ 3'exonuclease activity, and heat resistance. It means that the target property is maintained.
  • the polymerase active domain of the variant preferably consists of an amino acid sequence having 85% or more (preferably 90% or more or 95% or more) identity with the amino acid sequence of the polymerase active domain of the original DNA polymerase.
  • the mutant domain E preferably consists of an amino acid sequence having 85% or more (preferably 90% or more or 95% or more) identity with the amino acid sequence of domain E of the original DNA polymerase. Amino acid mutations in variants are preferably conservative substitutions.
  • the DNA polymerase is preferably a DNA polymerase belonging to Family A.
  • Examples of the DNA polymerase belonging to Family A include, but are not limited to, Taq polymerase, Tth polymerase, Z05 polymerase, Tma polymerase, Bca polymerase, Bst polymerase and the like.
  • the DNA polymerase is preferably at least one selected from the group consisting of Taq polymerase, Tth polymerase, and Z05 polymerase. In certain embodiments, it is preferably two or more DNA polymerases selected from the group consisting of Taq polymerase, Tth polymerase, and Z05 polymerase, and preferably selected from the group consisting of Taq polymerase and Tth polymerase and Z05 polymerase. More preferably, it is a combination with at least one kind.
  • the polymerase activity of DNA polymerase is measured as follows. If the polymerase activity is strong, storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% (v / v) polyethylene glycol sorbitan monolaurate (Tween). Dilute the DNA polymerase solution with 20), 0.1% (v / v) octylphenyl-polyethylene glycol (Nonidet TM P40), 50% (v / v) glycerin) and then do the following: Make a measurement.
  • the radioactivity of the filter is measured with a liquid scintillation counter (manufactured by Packard), and the uptake of nucleotides in the template DNA is measured.
  • One unit of polymerase activity is the amount of DNA polymerase that incorporates 10 nmol nucleotides per 30 minutes into the acid-insoluble fraction (that is, the fraction that insolubilizes when solution D is added) under these conditions.
  • Solution A 40 mM Tris-HCl buffer (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 ⁇ g / mL BSA (bovine serum albumin)
  • Solution B 1.5 ⁇ g / ⁇ l
  • C 1.5 mM dNTP (250 cpm / pmol [3H] dTTP)
  • Liquid D 20% (w / v) trichloroacetic acid (2 mM sodium pyrophosphate)
  • E 1 mg / mL calf thymus DNA
  • the antibody or fragment thereof of the present invention is SEQ ID NO: 1: Amino acid sequence shown in (Amino acid sequence of E domain of Taq polymerase (wild type)), SEQ ID NO: 2: Amino acid sequence shown in (Amino acid sequence of E domain of Tth polymerase (wild type)), SEQ ID NO: 3: (Preferably 85% or more, 90% or more, or 95% or more) identity with respect to the amino acid sequence shown in (Z05 polymerase (wild type) E domain amino acid sequence) and these amino acid sequences. It is preferable to bind to at least one E domain selected from the group consisting of the amino acid sequence having.
  • the antibody or fragment thereof of the present invention comprises at least a part or all of an amino acid region of any of the four amino acid regions enclosed in a square in the amino acid sequence represented by any of SEQ ID NOs: 1 to 3 above. It preferably binds (or recognizes) as one (eg, one, two, three, four, or five) epitopes.
  • amino acid region A the 56-66th amino acid region from the N-terminal of SEQ ID NO: 1 or the 56-67th amino acid region from the N-terminal of SEQ ID NO: 2 or 3 is referred to as "amino acid region A".
  • amino acid region B The 75-81st amino acid region from the N-terminal of SEQ ID NO: 1 or the 76-82th amino acid region from the N-terminal of SEQ ID NO: 2 or 3 is referred to as "amino acid region B"; from the N-terminal of SEQ ID NO: 1.
  • amino acid region C The 161 to 182nd amino acid region or the 162 to 183th amino acid region from the N end of SEQ ID NO: 2 or 3 is referred to as "amino acid region C"; the 269 to 285th amino acid region or SEQ ID NO: from the N terminal of SEQ ID NO: 1.
  • amino acid region D The 271 to 287th amino acid regions of 2 or 3 are referred to as "amino acid region D".
  • the polI polymerase family including Taq polymerase has a plurality of regions having particularly high conservability from the N-terminal to the vicinity of the 200th position (Kim Y et al., Mol. Cells, Vol. 7, No. 4, pp. 468-472 (the whole of which is incorporated herein by reference)).
  • the antibody or fragment thereof of the present invention is at least one (for example, one, two, three, four) present in the region (for example, amino acid regions A to C) near the 200th position from the N-terminal of the polI polymerase. , Or a fragment thereof that binds to the epitope of 5) is particularly preferable.
  • the antibody or fragment thereof of the present invention binds a part or all of the amino acid region A and / or the amino acid region B as at least one epitope.
  • an antibody or fragment thereof that binds at least a part or all (particularly all) of amino acid region B and a part or all (particularly part) of amino acid region C or D as an epitope is preferable.
  • the epitope of a part or all of the amino acid region A examples include, but are limited to, EDGDAVIVVF (SEQ ID NO: 60), KEDGDAVIVVF (SEQ ID NO: 61), EDGYKAVFVVF (SEQ ID NO: 62), and KEDGYKAVFVVF (SEQ ID NO: 63). Not done.
  • the epitope of some or all of the amino acid region A preferably comprises SEQ ID NO: 60 or 62.
  • the epitope of some or all of the amino acid region A is SEQ ID NO: 60, 61, or 62, more preferably SEQ ID NO: 61 or 62.
  • Examples of the epitope of a part or all of the amino acid region B include, but are not limited to, HEAYGGY (SEQ ID NO: 64) and HEAYEAS (SEQ ID NO: 65).
  • Examples of the epitope of a part or all of the amino acid region C include HLITPEWLW (SEQ ID NO: 66), KYGLRPEQWVDF (SEQ ID NO: 67), EKYGLRPDQWADY (SEQ ID NO: 68), KYGLRPDQWADY (SEQ ID NO: 69), GLRPEQWVDF (SEQ ID NO: 70).
  • the epitope of some or all of the amino acid region C preferably comprises SEQ ID NO: 68, 70, or 71. In a preferred embodiment, the epitope of some or all of the amino acid region C is SEQ ID NO: 66, 67, 68, 70, or 71.
  • Examples of the epitope of a part or all of the amino acid region D include LELLEF (SEQ ID NO: 75), LERREFGSLLH (SEQ ID NO: 76), LERREFGSLLHEF (SEQ ID NO: 77), LRAFLEXLEF (SEQ ID NO: 78), RAFLERLEF (SEQ ID NO: 79), and the like. Examples include, but are not limited to, RAFLELLEFGSLLH (SEQ ID NO: 80), LEFGSLLLH (SEQ ID NO: 81), LEFGSLHLHEF (SEQ ID NO: 82), and LRAFLEXLEFGSLLHEF (SEQ ID NO: 83).
  • the epitope of some or all of the amino acid region D preferably comprises SEQ ID NO: 75, 76, 78, 79, or 81. In a preferred embodiment, the epitope of some or all of the amino acid region D is SEQ ID NO: 77, 78, 80, or 82.
  • the length of the epitope is not particularly limited, but is preferably composed of, for example, 5 to 25 residues, preferably 6 to 20 residues, more preferably 6 to 15 residues, and 7 It is more preferably composed of up to 14 residues.
  • the heavy chain CDR1 of the antibody or fragment thereof of the present invention preferably comprises the following amino acid sequence: -Amino acid sequence represented by the formula (A-1) shown in Table 2.
  • XA3 is preferably T
  • XA4 is preferably F
  • XA5 is preferably D, N, or S
  • XA6 is preferably D, N, S. , K, or H, or D, N, S, or H
  • X A7 is preferably Y or W, or Y
  • X A8 is preferably G, W, or Y.
  • the heavy chain CDR1 of the antibody of the present invention or a fragment thereof is preferably from the amino acid sequence represented by the formula (A-1-1), more preferably from the formulas (A-1-2) to (A-1-9). It consists of an amino acid sequence selected from the group, or an amino acid sequence having 90% or more identity with respect to the amino acid sequence.
  • the identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the heavy chain CDR2 of the antibody or fragment thereof of the present invention preferably comprises the following amino acid sequence: -Amino acid sequence represented by the formula (B-1) shown in Table 3 An amino acid sequence having 90% or more (preferably 95% or more) identity with respect to the amino acid sequence, or 1 to 3 (preferably 1 or 2 or even more preferably 1) of the amino acid sequence. An amino acid sequence in which an amino acid is mutated (preferably a conservative substitution).
  • X B2 is preferably G, S, T, K, or N, G, S, or K, or S
  • X B3 is preferably Y, L, G, T, Or N, more preferably L, G, or N, L or N, or Y, G, or T
  • X B4 is preferably G, S, T, D, or H, even more preferably G, S. , T, or D, S or T, or G, D, or H
  • X B5 is preferably G or S, or G
  • X B6 is preferably G, S, T, or D.
  • X B7 is preferably S, T, Y, D, or H, more preferably S, T, or Y, S or T, or T, Y. , D, or H
  • X B8 is preferably S, T, V, I, or M, more preferably S, T, or I, S or T, or T, V, I, or M. ..
  • the heavy chain CDR2 of the antibody of the present invention or a fragment thereof is preferably from the amino acid sequence represented by the formula (B-1-1), more preferably from the formulas (B-1-2) to (B-1-10). It consists of an amino acid sequence selected from the group, or an amino acid sequence having 90% or more identity with respect to the amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the heavy chain CDR3 of the antibody or fragment thereof of the present invention preferably comprises the following amino acid sequence: Amino acid sequence represented by any of the formulas (C-1) to (C-6) shown in Table 4. An amino acid sequence having 90% or more (preferably 95% or more) identity with respect to the amino acid sequence, or 1 to 3 (preferably 1 or 2 or even more preferably 1) of the amino acid sequence. An amino acid sequence in which an amino acid is mutated (preferably a conservative substitution).
  • X C1 is preferably A or R, more preferably R, X C2 is preferably P or R, even more preferably R, and X C3 is preferably T or I, further. It is preferably T, X C4 is preferably V or L, more preferably V, X C5 is preferably P or A, even more preferably P, and X C6 is preferably T or Y, even more preferably. T, X C7 is preferably V, T, or N, more preferably V.
  • X C1 is preferably A or R, more preferably A, X C2 is preferably P or R, even more preferably P, and X C3 is preferably T or I, further. It is preferably I, X C4 is preferably V or L, X C5 is preferably P or A, more preferably A, X C6 is preferably T or Y, still more preferably Y, X. C7 is preferably V, T, or N, more preferably V or T.
  • X C7 is preferably V, T, or N, more preferably N.
  • X C7 is preferably V, T, or N, more preferably V.
  • X C7 is preferably V, T, or N, more preferably V.
  • the heavy chain CDR3 of the antibody or fragment thereof of the present invention preferably has the formulas (C-1-1), (C-2-1), (C-3-1), (C-4), (C-5).
  • the light chain CDR1 of the antibody or fragment thereof of the present invention preferably comprises the following amino acid sequence: -Amino acid sequence represented by the formula (D-1) shown in Table 5.
  • X D1 is preferably Q
  • X D2 is preferably G or S
  • X D3 is preferably V or I
  • X D4 is preferably S or K
  • X D5 is preferably S, N, or K, or S or N
  • X D6 is preferably F, Y, or Y.
  • the light chain CDR1 of the antibody of the present invention or a fragment thereof preferably comprises an amino acid sequence represented by the formula (D-1-1), more preferably (D-1-2) to (D-1-8). It consists of an amino acid sequence selected from the group or an amino acid sequence having 90% or more identity with respect to the amino acid sequence.
  • the identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the light chain CDR2 of the antibody or fragment thereof of the present invention preferably comprises the following amino acid sequence: Amino acid sequence represented by the formula (E-1) shown in Table 6A, An amino acid sequence having 90% or more (preferably 95% or more) identity with respect to the amino acid sequence, or 1 to 3 (preferably 1 or 2 or even more preferably 1) of the amino acid sequence. An amino acid sequence in which an amino acid is mutated (preferably a conservative substitution).
  • XE1 is preferably G, T, D, Y, or R, more preferably G, Y, or R, or G, T, D, or R, and even more preferably Y, Alternatively, it is G, T, D, or R, X E2 is preferably A, T, or V, more preferably A or T, or A or V, and X E3 is preferably K, D, N. , Or S, more preferably K, D, or N, or K or S, even more preferably D or N, or K or S.
  • the light chain CDR2 of the antibody of the present invention or a fragment thereof is preferably represented by the amino acid sequence represented by the formula (E-1-1), more preferably represented by (E-1-2) or (E-1-3).
  • Amino acid sequence to be used more preferably an amino acid sequence selected from the group consisting of (E-1-4) to (E-1-12), or an amino acid sequence having 90% or more identity with respect to the amino acid sequence. Consists of.
  • the identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • the C-terminus of the light chain CDR2 of the antibody or fragment thereof of the present invention is preferably adjacent to the following amino acid sequence: Amino acid sequence represented by the formula (E-2) shown in Table 6B, An amino acid sequence having 90% or more (preferably 95% or more) identity with respect to the amino acid sequence, or 1 to 3 (preferably 1 or 2 or even more preferably 1) of the amino acid sequence. An amino acid sequence in which an amino acid is mutated (preferably a conservative substitution).
  • X E4 is preferably S, R, N, Y, or T, more preferably S, or R, N, Y, or T
  • X E5 is preferably L or R
  • X E6 is preferably A, P, or Y, more preferably A or P, or A or Y
  • X E7 is preferably S or T, or S.
  • the amino acid sequence represented by the formula (E-2-1) or (E-2-2) is preferably attached to the C-terminal of the light chain CDR2 of the antibody of the present invention or a fragment thereof, and more preferably (E-2-). 3) An amino acid sequence selected from the group consisting of (E-2-8) or an amino acid sequence having 90% or more identity with the amino acid sequence is adjacent. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • amino acid adjacent to the N-terminal of the light chain CDR2 of the antibody or fragment thereof of the present invention is not particularly limited, but is preferably Y, F, or H, and it is also preferable that these amino acids are conservatively substituted. ..
  • the light chain CDR3 of the antibody or fragment thereof of the present invention preferably comprises the following amino acid sequence: -Amino acid sequence represented by the formula (F-1) or (F-2) shown in Table 7.
  • X F1 is preferably L, Q, F, or Y, more preferably L, Q, or Y, or Q, F, or Y, and even more preferably L, Q, or Y.
  • Q or F, X F2 is preferably Q
  • X F3 is preferably S or Y, or Y
  • X F4 is preferably G, N, Q, or Y, even more preferably.
  • N preferably G, S, Y, or W, more preferably G, Y, or W; S, Y, or W; or G or S, and X F7 is preferably S. , P, or W, more preferably P or W; S or P; or P, and XF8 is preferably L, P, H, or Y, more preferably L, H, Y, or T, or. It is P.
  • the light chain CDR3 of the antibody of the present invention or a fragment thereof is preferably an amino acid sequence represented by the formula (F-1-1) or (F-2-1), and more preferably (F-1-2) to ( It consists of an amino acid sequence represented by F-1-5) and (F-2-2) to (F-2-4), or an amino acid sequence having 90% or more identity with respect to the amino acid sequence.
  • the identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.
  • Table 8A shows suitable examples of combinations of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 of the antibody or fragment thereof of the present invention.
  • Table 8B shows a suitable example of the combination of the sequences adjacent to the C-terminal of the heavy chain CDR1 to 3, the light chain CDR1 to 3, and the light chain CDR2 of the antibody or fragment thereof of the present invention.
  • 1 to 3 in at least one amino acid sequence among the sequences adjacent to the C-terminus of heavy chain CDR1 to 3, light chain CDR1 to 3, and light chain CDR2. , More preferably one) amino acid is mutated (preferably conservative substitution).
  • the regions other than the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 are not particularly limited as long as they can be used for an antibody, and can be any amino acid sequence.
  • the constant region for example, the constant regions of IgG1, IgG2, IgG3, IgA1, IgA2, IgM and the like can be used, but the constant region is not limited thereto.
  • the constant region may be a constant region derived from any animal such as a mammal such as mouse, hamster, rat, guinea pig, rabbit, ferret, goat, monkey or human, and may be, for example, SEQ ID NOs: 51 and 52.
  • Amino acid sequence shown in (heavy chain and light chain constant region derived from guinea pig), amino acid sequence shown in SEQ ID NOs: 53 and 54 (heavy chain and light chain constant region derived from mouse), 80% of these amino acid sequences.
  • Examples thereof include a sequence region consisting of an amino acid sequence having the above (preferably 85% or more, 90% or more, or 95% or more) identity.
  • the equilibrium dissociation constant (KD) of the antibody of the present invention or a fragment thereof with respect to domain E of the DNA polymerase is, for example, 50 nM or less, preferably 10 nM or less, for example, 1 pM or more.
  • KD can be measured using Biacore TM X100 ( Cytiva ), for example, as in the examples described below.
  • CM5 sensor chip Cytiva
  • a flow cell in which a ligand (DNA polymerase having a 5' ⁇ 3'exonuclease active domain) is immobilized and blocking is performed with a 1M ethanolamine hydrochloride solution to immobilize the ligand is prepared, and then a step-diluted antibody is applied to the flow cell.
  • KD can be calculated by adding above and analyzing the reaction signal.
  • the ability of the DNA polymerase to inhibit the 5' ⁇ 3'exonuclease activity is, for example, 50%.
  • the above is preferably 60% or more, 70% or more, 80% or more, or 90% or more.
  • the inhibitory ability is the following component: as in the examples described later.
  • Substrate nucleic acid ⁇ DNA labeled with radioisotopes DNA polymerase alone (1 unit (U)) or DNA polymerase (1U) and the antibody of the invention or a fragment thereof (0.005 ⁇ g / ⁇ L); 10 mM Tris-HCl (pH 8.6); 50 mM KCl; and 1.5 mM MgCl 2
  • the radioactivity of the labeled base released when the solution containing the above is incubated at 37 ° C. for 24 hours can be measured and calculated by the following formula.
  • N1 The amount of free nucleotides before incubating a solution containing no antibody of the present invention or a fragment thereof at 37 ° C. for 24 hours (or after incubating at ⁇ 20 ° C. for 24 hours), or the antibody of the present invention or a fragment thereof.
  • N2 Amount of free nucleotides after incubating a solution without the antibody of the present invention or a fragment thereof at 37 ° C. for 24 hours
  • N3 This Amount of free nucleotides after incubating a solution containing the antibody of the invention or a fragment thereof at 37 ° C. for 24 hours.
  • the substrate DNA (where the substrate DNA may be single-stranded or double-stranded, the substrate DNA is a probe) at 25 ° C. for 24 hours.
  • DNA polymerase which may have the function of DNA
  • the ability of the DNA polymerase to inhibit the 5' ⁇ 3'exonuclease activity is, for example, the following (1).
  • (2) can be confirmed quantitatively.
  • reaction solution in (1) and (2) examples include, for example.
  • IL6 Interleukin 6, Assay ID: Hs0985369_m1 (hereinafter abbreviated as "IL6”), Cyclin-dependent kinase 10, Assay ID: Hs00177586_m1 (hereinafter abbreviated as "CDK10”), APC, WNT signaling pathway regulator, Assay ID: Hs01568269_m1 (hereinafter abbreviated as "APC”), mitogen-activated protein kinase 8, Assay ID: Hs00177083_m1 (hereinafter abbreviated as "MAPK8”), SIVA1 apoptosis inducing factor, Assay ID: Hs00276002_m1 (hereinafter abbreviated as "SIVA1”), Ribosomal protein S19, Assay ID: Hs0
  • F 11 Fluorescence intensity at the beginning of the cycle before exposure to 24 hours at 25 ° C (or after exposure to -20 ° C for 24 hours) without the antibody of the present invention or a fragment thereof
  • F 12 The antibody or fragment thereof of the present invention Fluorescence intensity at the beginning of the cycle after exposure to 24 hours at 25 ° C without inclusion
  • F 13 Fluorescence intensity at the beginning of the cycle after exposure to 24 hours at 25 ° C with the antibody of the present invention or a fragment thereof
  • the value (Ct value ratio) in the above (a) is preferably 0.9 or more.
  • the value (fluorescence intensity ratio) in the above (b) is preferably 0.35 or more, more preferably 0.5 or more, and further preferably 0.7 or more.
  • the value (fluorescent-labeled substrate DNA decomposition rate) in the above (c) is preferably 30% or less, more preferably 20% or less.
  • the substrate DNA at 25 ° C. or 37 ° C. for 24 hours (where the substrate DNA may be single-stranded or double-stranded.
  • the substrate DNA may have a function as a probe
  • the inhibitory ability of the DNA polymerase to the 5' ⁇ 3'exonuclease activity is, for example.
  • the band strength of the substrate DNA is compared by gel electrophoresis for the solution after 24 hours exposure at ⁇ 20 ° C. or before exposure at 25 ° C. for 24 hours).
  • the smaller the difference in band intensity the higher the ability of DNA polymerase to inhibit the 5' ⁇ 3'exonuclease activity.
  • Fluorescence intensity or spectrophotometer at the beginning of the cycle by real-time PCR for the reaction solution compare the fluorescence intensities in. The smaller the difference in fluorescence intensity, the higher the ability of DNA polymerase to inhibit the 5' ⁇ 3'exonuclease activity.
  • Examples of the solution or reaction solution in (3) and (4) include, for example.
  • the substrate DNA may or may not be labeled with a fluorescent dye, a radioisotope, or the like.
  • the substrate DNAs as an example of the double-stranded substrate DNA, the 3'end of at least one strand is more prominent than the 5'end of the other strand, as represented by the combination of SEQ ID NO: 56 and SEQ ID NO: 57.
  • the main-strand substrate DNA may be mentioned, but is not particularly limited.
  • the base length of the protruding portion is, for example, about 3 to 10 base lengths.
  • examples of the gel electrophoresis method include, but are not limited to, agarose gel electrophoresis, polyacrylamide gel electrophoresis, and the like.
  • a device capable of quantifying the band strength of nucleic acid examples include a microchip potential electrophoresis device for DNA / RNA analysis (MultiNA, Shimadzu Corporation) or a fully automated high-throughput electrophoresis system (TapeStation). Series, Agilent Technologies Co., Ltd.), etc., but are not particularly limited.
  • the substrate DNA is fluorescently labeled.
  • the double-stranded substrate DNA examples include a combination of SEQ ID NO: 58 and SEQ ID NO: 59, in which the 3'end of at least one strand protrudes from the 5'end of the other strand and A double-stranded substrate DNA in which at least one end of the other strand is fluorescently labeled can be mentioned, but is not particularly limited.
  • the base length of the protruding portion is, for example, about 3 to 10 base lengths.
  • the change in the fluorescence value may be, for example, measured by a real-time PCR device or a spectrophotometer, but is not particularly limited.
  • F 22 The antibody of the present invention or a fragment thereof Fluorescence intensity at the beginning of the cycle after exposure to 24 hours at 37 ° C without inclusion
  • F 23 Fluorescence intensity at the beginning of the cycle after exposure to 24 hours at 37 ° C with the antibody of the present invention or a fragment thereof.
  • the substrate DNA decomposition rate in the above (d) is preferably 30% or less, more preferably 20% or less.
  • the fluorescence-labeled substrate DNA decomposition rate in the above (e) is preferably 30% or less, more preferably 20% or less.
  • the antibody or fragment thereof of the present invention may be allowed to coexist with a nucleic acid such as a DNA polymerase and a nucleic acid template, a primer, a probe, for example, at 25 ° C. for 24 hours, 48 hours, or 72 hours, or at 37 ° C. for 24 hours. , Nucleic acid degradation due to 5' ⁇ 3'exonuclease activity of DNA polymerase can be suppressed. Therefore, the antibody of the present invention or a fragment thereof can be suitably used for improving the stability of a reagent for nucleic acid amplification or the like.
  • the antibody of the present invention or a fragment thereof can be obtained, for example, by immunizing an animal with a part or all of the DNA polymerase containing domain E as an immunogen.
  • the immunogen is preferably a portion containing domain E of DNA polymerase, more preferably a portion containing domain E of at least one DNA polymerase selected from the group consisting of Taq polymerase, Tth polymerase, and Z05 polymerase. Yes, more preferably a portion of the Tth polymerase comprising domain E.
  • Animals include, but are not limited to, mammals such as mice, hamsters, rats, guinea pigs, rabbits, ferrets, goats, monkeys and humans.
  • the antibody of the present invention can be obtained by screening an antibody produced by an animal immunized with the above-mentioned immunogen. For example, when a part of the DNA polymerase including the domain E is used as an immunogen, screening may be performed using the binding ability to all of the DNA polymerase as an index, and when the whole DNA polymerase is used as an immunogen, the domain of the DNA polymerase may be screened. You may screen using the difference between the binding ability to a part including E or the binding ability to all of the DNA polymerase and the binding ability to other than the part including the domain E of the DNA polymerase as an index.
  • Specific screening methods may be, for example, a hybridoma method in which mammalian spleen cells and myeloma cells are fused, a phage display method in which an antibody having an affinity for a target molecule is selected from an antibody phage library, or the like.
  • a method may be used in which antigen-specific plasma cells are selected from immune animals, an antibody gene (full length or a part of a variable region, etc.) is isolated, and a recombinant antibody having a high affinity for the antigen is obtained.
  • Methods for selecting antigen-specific plasma cells include, for example, the method described in US Patent Application Publication No. 2014/031528 (which is incorporated herein by reference in its entirety) and US Patent Application Publication No. 2018 /. Included is the method described in 292407, which is incorporated herein by reference in its entirety.
  • an antigen-specific plasma cell is identified by allowing a fluorescently labeled antigen and a follicle-affinitive fluorescent dye to act on a cell suspension solution prepared from an immune animal and fluorescently labeling the antibody expressed on the cell surface. can do.
  • a cell group containing antibody-producing cells is subjected to immobilization treatment using a cross-linking agent and cell membrane lysis treatment using a surfactant, and the antibody expressed inside the cells is bound to a fluorescently labeled antigen.
  • Antigen-specific plasma cells can be identified.
  • at least one plasma cell bound to the target antigen can be separated by performing single cell analysis using a cell sorter.
  • a fluorescent probe that has high staining selectivity for the endoplasmic reticulum of cells and can distinguish plasma cells and plasma blast cells from other cells can be used.
  • the fluorescent probe for example, the one described in US Patent Application Publication No. 2013/029325 (which is incorporated herein by reference in its entirety) can be used.
  • Examples of the method for obtaining an antibody gene from an antigen-specific plasma cell include, but are not limited to, a hybridoma method and cloning of an antibody gene. Examples of the latter method include a method of extracting mRNA from antigen-specific plasma cells, performing reverse transcription, and acquiring an antibody gene by synthesizing cDNA.
  • US Patent Application Publication No. 2011/020879 It may be the method described in (the whole thereof is incorporated in the specification by reference).
  • This method is a method of extracting mRNA from antigen-specific plasma cells using magnetic beads and acquiring an antibody gene by RT-PCR, including optional washing steps, cDNA synthesis from mRNA, DNA amplification, etc. This is a method using a reaction jig capable of carrying out a plurality of sequential reactions in parallel.
  • the method of obtaining a recombinant antibody from an antibody gene may be, for example, a method of preparing an antibody expression vector containing an antibody gene and expressing the antibody from this antibody expression vector.
  • Such methods include, for example, the method described in U.S. Patent Application Publication No. 2013/023009 (which is incorporated herein by reference in its entirety) and U.S. Patent Application Publication No. 2011/11769 (by reference).
  • the method described in (the whole of which is incorporated in the specification) can be mentioned.
  • the former method by linking one or more double-stranded DNA fragments to a PCR amplification product containing a target gene sequence, a sequence derived from the target gene is included without purifying the PCR amplification product.
  • Linked DNA fragments can be specifically prepared.
  • the target DNA fragment is selectively selected by retaining the homologous recombination region at both ends of the linearized vector with the sequence inside the amplification primer and the amplification primer sequence present only in the target gene.
  • the vector can be constructed by homologous recombination.
  • the antibody or fragment thereof of the present invention can also be obtained by a genetic engineering method based on the amino acid sequence information of the antibody or fragment thereof obtained by the above method.
  • the C-terminal adjacent regions of the light chain CDR1 to 3 and optionally the light chain CDR2 are for the amino acid sequences of the light chain CDR1 to 3 and optionally the C-terminal adjacent region of the light chain CDR2 of the antibody obtained by the above method, respectively. It was designed to have 80% or more identity and to have 80% or more identity for each of the heavy chain CDRs 1 to 3 with respect to the amino acid sequences of the heavy chain CDRs 1 to 3 of the antibody obtained by the above method.
  • An expression vector incorporating an antibody gene can also be obtained by expressing it in any host cell known in the art.
  • polynucleotide of the present invention preferably contains the coding sequence of the antibody or fragment thereof described in 2 above.
  • the polynucleotide of the present invention preferably contains an expression cassette of the antibody or fragment thereof described in 2 above.
  • the expression cassette is not particularly limited as long as it allows expression in the host cell, and includes, for example, a promoter and a coding sequence arranged under the control of the promoter.
  • the promoter is not particularly limited and can be appropriately selected according to the type of host cell.
  • various pol II promoters can be used.
  • the pol II promoter is not particularly limited, and examples thereof include a CMV promoter, an EF1 promoter, an SV40 promoter, and an MSCV promoter.
  • examples of the promoter include tryptophan promoters such as trc and tac; lac promoter; T7 promoter; T5 promoter; T3 promoter; SP6 promoter; arabinose-inducible promoter; cold shock promoter; tetracycline-inducible promoter and the like.
  • the expression cassette may contain other elements, if necessary.
  • Other elements include, for example, a multicloning site (MCS), drug resistance gene, origin of replication, enhancer sequence, repressor sequence, insulator sequence, reporter protein coding sequence, drug resistance gene coding sequence and the like. These may be one kind alone or a combination of two or more kinds.
  • MCS multicloning site
  • the polynucleotide of the present invention can be, for example, in the form of a vector.
  • An appropriate vector is selected according to the purpose of use, the type of host cell, and the like.
  • E. coli-hosted vectors include M13 phage or a variant thereof, ⁇ phage or a variant thereof, pBR322 or a variant thereof (eg, pB325, pAT153, pUC8), and yeast-hosted vectors include pYepSec1, pMFa.
  • vectors such as pYES2 and pPIC3.5K are pAc and pVL as vectors having insect cells as hosts, and pcDNA, pCDM8, pMT2PC and the like can be exemplified as vectors using mammalian cells as hosts.
  • the cell of the present invention preferably contains the polynucleotide described in 3 above.
  • the cells include, for example, Escherichia coli K12 and other Escherichia coli, Bacillus subtilis MI114 and other Bacillus spp. Examples include cells, animal cells and the like.
  • the animal cells are preferably cultured cells derived from mammals, specifically, COS7 cells, CHO cells, HEK293 cells, Expi293 cells, 293F cells, 293T cells, 293FT cells, Hela cells, PC12 cells, N1E-115 cells. , SH-SY5Y cells and the like.
  • the cells of the invention preferably express an antibody or fragment thereof that specifically binds to domain E of the DNA polymerase.
  • the cells of the invention preferably secrete or have on the cell surface an antibody or fragment thereof that specifically binds to domain E of the DNA polymerase.
  • the reagent of the present invention preferably contains the antibody or fragment thereof described in 2 above, the polynucleotide described in 3 above, or the cell described in 4 above.
  • the reagent of the present invention preferably further contains an excipient or carrier and / or an additive.
  • excipient or carrier examples include starch, lactose, crystalline cellulose, sorbitol, calcium hydrogen phosphate, water, ethanol, (poly) ethylene glycol, (poly) propylene glycol, glycerol, vegetable oil and the like. These can be used alone or in combination of two or more.
  • the additive examples include a buffering agent, an tonicity agent, a thickener, a chelating agent, an emulsifier, a coloring agent, a preservative and the like. These can be used alone or in combination of two or more.
  • the reagent of the present invention is preferably a reagent for nucleic acid amplification.
  • the reagent of the present invention preferably contains a DNA polymerase having domain E, and an antibody or a fragment thereof that specifically binds to domain E of the DNA polymerase.
  • the molar ratio of the antibody or fragment thereof to the DNA polymerase is not limited as long as the effect of the present invention is obtained, but it is preferably about 1: 1 to about 500: 1.
  • the reagent may further contain a DNA polymerase that does not have domain E.
  • the reagent is preferably a nucleic acid amplification reagent.
  • the reagents of the invention are at least one selected from the group consisting of a DNA polymerase having domain E, a primer, a probe, and a deoxyribonucleoside-5'-phosphate, and domain E of the DNA polymerase (preferably).
  • the reagent may further contain, for example, a metal salt such as manganese or magnesium, a buffer, or the like in order to improve the DNA polymerase activity.
  • the reagent is preferably a nucleic acid amplification reagent.
  • the reagent of the present invention contains a DNA polymerase having domain E
  • examples of the DNA polymerase include those described in 2 above.
  • the primer may be at least two kinds of primers.
  • the at least two primers are oligonucleotides that are substantially complementary to the nucleic acid sequence to be amplified, and define both ends of the nucleic acid sequence to be amplified, and the extension product synthesized from each primer. When separated from its complement, it can serve as a template for further synthesis.
  • the primer can be appropriately selected, designed and used according to the target nucleic acid, and is not particularly limited. Furthermore, if the target nucleic acid to be targeted is assumed to be a subtype, it may be a degenerate primer. In general, the primer can be an oligonucleotide having 12-60 nucleotides. Primers can be synthesized by a DNA synthesizer or isolated from a biological source.
  • the probe may be a hybridization probe labeled with at least one labeling substance.
  • the analysis of the nucleic acid amplification product can be monitored by monitoring the fluorescent signal instead of the usual electrophoresis, and the analysis labor is reduced. Furthermore, it is not necessary to open the reaction vessel, and the risk of contamination can be further reduced.
  • the hybridization probe include TaqMan hydrolysis probe [US Pat. No. 5,210,015, US Pat. No. 5,538,848, US Pat. No.
  • the reagent of the present invention may contain a double-stranded DNA-bound fluorescent compound instead of the probe.
  • the double-stranded DNA-bound fluorescent compound include SYBR (registered trademark) Green I, SYBR (registered trademark) Gold, SYTO-9, SYTP-13, SYTO-82 (Life Technologies), and EvaGreen (registered trademark; Biotium). , LCGreen (Idaho), LightCycler (registered trademark) 480 ResoLight (Roche Applied Science) and the like, but are not limited thereto.
  • the deoxyribonucleoside-5'-phosphoric acid is, for example, dATP, dCTP, dTTP, dGTP, or a mixture thereof.
  • dATP include those that have been chemically modified.
  • the nucleic acid amplification method includes, for example, PCR method, Loop-Mediated Isothermal Amplification (LAMP) method, Transcriction Reflection Transcriction Transition Constraint Reaction (TRC) method, Nucleic acid amplification method, Nucleic acid amplification method, Nucleic acid amplification method, and Nucleic acid amplification method. (NASBA) method and the like can be mentioned, but the method is not limited thereto.
  • the nucleic acid amplification method is preferably the PCR method.
  • PCR methods for example, a PCR method in which primer annealing is inhibited up to a predetermined temperature with a monoclonal antibody specific for DNA polymerase, a so-called hot start PCR method, is preferable.
  • the hot start PCR reagent of the present invention further effectively causes a non-specific reaction by containing a combination of an antibody that specifically binds to the polymerase active domain of DNA polymerase and an antibody that specifically binds to domain E of DNA polymerase. Can be suppressed.
  • the hot start PCR reagent of the present invention specifically binds to a primer, deoxyribonucleoside-5'-phosphate, DNA polymerase, an antibody that specifically binds to the polymerase active domain of DNA polymerase, and domain E of DNA polymerase. It preferably contains an antibody.
  • the reagent is mixed with a reagent containing a target nucleic acid, and the obtained mixture is heated to 60 ° C. or higher (for example, heated at 95 ° C. for 20 seconds or longer) to inactivate both antibodies to generate primer extension. Objects can be formed.
  • Test example 1 Preparation of antigen
  • Taq polymerase having the amino acid sequence of SEQ ID NO: 49 TAP-201, Toyo Spinning Co., Ltd., hereinafter referred to as "wole Taq”
  • SEQ ID NO: 50 SEQ ID NO: 50
  • Tth polymerase having an amino acid sequence THH-301, Toyobo Co., Ltd., hereinafter referred to as "where Tth" was used.
  • Tth Tth polymerase having an amino acid sequence
  • Tth was used.
  • the sequence identity of whole Taq and whole Tth is about 87%.
  • Tth exo a polypeptide having the amino acid sequence of SEQ ID NO: 1 (from the N-terminal to the 290th amino acid of whole Taq) (hereinafter referred to as "Taq exo"), and A polypeptide having the amino acid sequence of SEQ ID NO: 2 (from the N-terminal to the 292nd amino acid of whole Tth) (hereinafter referred to as "Tth exo”) is designated as E.I. It was expressed using the coli JM109 strain, purified using heparin sepharose chromatography, and used. Both antigens were dissolved in phosphate buffer.
  • Test example 2 Guinea pig immunity Slc: A Hartley guinea pig (7-week-old male) was injected subcutaneously (lumbar) into the back subcutaneously (lumbar) with 0.8 mL of an antigen preparation of 400 ⁇ g of antigen.
  • the antigen preparation the antigen solution in which the antigen was dissolved in the phosphate buffer in Test Example 1 and the adjuvant TitterMAX Gold (TiterMAX) were mixed at a ratio of 1: 1 (liquid volume ratio) and emulsified. After 3 weeks, an additional 0.8 mL of antigen preparation of 400 ⁇ g of antigen was injected for booster immunization.
  • Test example 3 Preparation of Fluorescently Labeled Protein
  • the entire DNA polymerase and the DNA polymerase lacking domain E were fluorescently labeled.
  • the domain E-deficient DNA polymerases are Taq polymerase (hereinafter referred to as “ ⁇ Taq”) in which the amino acids from the N-terminal to the 289th amino acid are deleted in SEQ ID NO: 49, and the 291st from the N-terminal in SEQ ID NO: 50.
  • Tth polymerases (hereinafter referred to as “ ⁇ Tth”) lacking the amino acids up to E. It was expressed using the coli JM109 strain and purified using heparin sepharose chromatography.
  • Test example 4 Isolation of Domain E-Specific Plasma Cells and Construction of Antibody Expression Vectors by the methods described in US Patent Application Publication No. 2014/031528, US Patent Application Publication No. 2018/292407, and US Patent Application Publication No. 2013/0292325.
  • a cell suspension was prepared from the iliac lymph node from the guinea pig immunized in Test Example 2, and domain E-specific plasma cells were selected using a flow cytometer. Domain E-specific plasma cells were selected by the following five methods by changing the combination of the antigen used for immunization and the fluorescently labeled protein prepared in Test Example 3.
  • the number of plasma cells selected targeting domain E of Taq polymerase was larger in method 3 than in methods 1 and 2, and was 288 in method 3.
  • the number of plasma cells selected targeting domain E of Tth polymerase was 192 in method 4 and 240 in method 5.
  • guinea pig lymph node swelling was larger and the number of isolated plasma cells was larger than in methods 1 and 2, so Tth polymerase was an immune response as an antigen compared to Taq polymerase. Can be said to have been large. Therefore, it was found that by using Tth polymerase as an antigen, domain E-specific plasma cells can be efficiently selected for both Taq polymerase and Tth polymerase. Further, in the method 5, the number of isolated plasma cells and the antibody expression vector were larger than those in the method 4. Therefore, it was found that an antibody (anti-domain E antibody) that specifically binds to domain E can be obtained more efficiently when immunized only with domain E than with immunization with whole DNA polymerase.
  • Test example 5 Evaluation of binding ability of antibody to domain E
  • the antibody expression vector was introduced into 293FT cells by the method described in US Patent Application Publication No. 2018/292407, and the culture supernatant in which the antibody was secreted was collected.
  • a commercially available hot start antibody (TCP-101, manufactured by Toyobo Co., Ltd.) was immobilized on an ELISA plate (Sumitomo Bakelite, MS-8896F) using a carbonate buffer. After washing each well, blocking was performed using 1 ⁇ TBS (Nacalai Tesque) containing 1% (w / v) bovine serum albumin (globulin-free, Nacalai Tesque).
  • an antigen (wole Taq, whole Tth) diluted with 1 ⁇ TBS-T (Nacalai Tesque) was added to each well. After washing each well, the culture supernatant was added to each well. After washing each well, Goat Anti-Guinea pig IgG H & L (HRP) (Abcam) was added after diluting it 50,000 times. After washing each well, a TMB solution (TMBW-1000-01, SURMODICS) was added to develop color, 1N sulfuric acid (Nacalai Tesque) was added to stop the reaction, and then a plate reader was used to stop the reaction at 450 to 620 nm. The wavelength of was measured. In this binding ability evaluation, since the DNA polymerase active domain is occupied by the immobilized antibody, the binding ability of the antibody to the domain E is evaluated.
  • the hit rate for each DNA polymerase and the hit rate for both DNA polymerases were about 2 to 8 times higher than those in method 4 and method 5. From this result, in the acquisition of an antibody that specifically binds to domain E of Taq polymerase, a method for selecting domain E-specific plasma cells using Tth Exo, which strongly induces an immune response, as an antigen and fluorescently labeled whole Taq. Can be said to be efficient. Also, in the acquisition of an antibody that specifically binds to domain E of Tth polymerase, a method of selecting domain E-specific plasma cells using Tth Exo as an antigen and fluorescently labeled whole Tth produces an unexpected effect. It has been found that domain E-specific antibodies can be isolated with high probability.
  • Test Example 6 Degradation of probe by DNA polymerase having domain E It was confirmed that the probe was degraded when the PCR reaction solution containing the DNA polymerase having domain E was exposed at 25 ° C. for 24 hours.
  • PCR reaction solution [Mix for PCR] A PCR mix 1 having the composition shown below was prepared.
  • PCR Mix 1 Taq polymerase (0.05U / ⁇ L, TAP-201, manufactured by Toyobo Co., Ltd.); Anti-polymerase antibody for hot start PCR (0.01 ⁇ g / ⁇ L, TCP-101, Made by Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.5 mM MgCl 2 ; and 0.3 mM dNTPs.
  • CDNA prepared from HeLa cell (derived from human cervical cancer) RNA was used.
  • Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep TM II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) were used for RNA extraction and cDNA synthesis. I followed the book.
  • each gene is detected on the FAM channel.
  • Table 9 shows the Ct values when each gene (IL6, CDK10, APC, MAPK8, SIVA1, RPS19, SERPINB5) in HeLa cDNA was detected using real-time PCR, and the fluorescence value at the 10th cycle in Multicomponent Data. ..
  • the fluorescence values at the 10th cycle increased for all 7 types of genes as compared with the case where the PCR reaction solution was exposed at -20 ° C for 24 hours.
  • the reason for the increase in fluorescence value is that exposure at 25 ° C. for 24 hours caused the fluorescently labeled probe to be degraded before the start of the cycle, releasing the fluorescent label, resulting in dequenching by the quencher and fluorescence. It is presumed. Therefore, it was found that all fluorescently labeled probes that detect 7 kinds of genes are degraded when exposed at 25 ° C. for 24 hours.
  • Test Example 7 Suppression of probe degradation by anti -domain E antibody When a PCR reaction solution containing a DNA polymerase having domain E and an anti-domain E antibody is exposed at 25 ° C. for 24 hours, it is confirmed whether the anti-domain E antibody suppresses probe degradation. did.
  • PCR Mix 3 Tth polymerase (mutant) (0.05 U / ⁇ L) according to WO2018 / 096961; Anti-polymerase antibody for hot start PCR (0.01 ⁇ g / ⁇ L, TCP-101, manufactured by Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.3); 80 mM KCl; 1.5 mM MgCl 2 ; 0.5 mg / mL BSA; 0.1% (v / v) TritonX-100; 0.1% (w / v) sodium cholic acid; and 0.3 mM dNTPs.
  • CDNA prepared from HeLa cell (derived from human cervical cancer) RNA was used.
  • Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep TM II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) were used for RNA extraction and cDNA synthesis. I followed the book.
  • reaction solution 1 Primer / probe was mixed at a ratio of 1/20 and nucleic acid template at a ratio of 1/20 to Mix 1 for PCR to prepare 19 ⁇ L of the mixed solution.
  • 1 ⁇ L of 20 mM Tris-HCl (pH 7.5) was added to the mixture and exposed at ⁇ 20 ° C. or 25 ° C. for 24 hours.
  • 1 ⁇ L of Platinum Taq Monoclonal Antibody (10965-028, Thermo Fisher Scientific) was added to the mixed solution and exposed at 25 ° C. for 24 hours.
  • reaction solution 2 For the anti-domain E antibody, 1 ⁇ L of each 0.8 mg / mL solution was added to the mixed solution (carry-on amount: 0.8 ⁇ g), and the mixture was exposed at 25 ° C. for 24 hours.
  • Reaction solution 2 Primer / probe was mixed at a ratio of 1/20 and nucleic acid template at a ratio of 1/20 to Mix 2 for PCR to prepare 19 ⁇ L of the mixed solution.
  • 1 ⁇ L of 20 mM Tris-HCl (pH 7.5) was added to the mixture and exposed at ⁇ 20 ° C. or 25 ° C. for 24 hours.
  • reaction For the anti-domain E antibody, 1 ⁇ L of each 1.2 mg / mL solution was added to the mixed solution (carry-on amount: 1.2 ⁇ g), and the mixture was exposed at 25 ° C. for 24 hours.
  • reaction Reactions 1 to 3 were reacted in the following temperature cycle using a real-time PCR device (Applied Biosystems 7500 Fast real-time PCR system). The fluorescence value was read in an extension step at 60 ° C. for 60 seconds.
  • Tempoture cycle Step 1: 95 ° C 1 minute
  • Step 2 95 ° C 15 seconds-60 ° C 60 seconds 50 cycles (PCR)
  • the Ct value of the RPS19 gene was delayed by about 5 to 6 as compared with the case where it was exposed at -20 ° C. for 24 hours. It can be said that the sensitivity is reduced by about 25 to 26 times. On the other hand, in the reaction solution 1 to which the anti-domain E antibody was added, the Ct value was all lower than 30.
  • the cause is that the reaction solution reached room temperature when the control reagent was prepared or set in the real-time PCR device, and the fluorescently labeled probe was decomposed. Therefore, this antibody can suppress the decomposition of the probe in the reaction solution not only during long-term storage of the reaction solution but also during preparation of a normal nucleic acid amplification reagent.
  • Test Example 8 Effect of exposure time of PCR reaction solution at 25 ° C The effect of suppressing probe degradation of anti-domain E antibody was confirmed by changing the exposure time of PCR reaction solution at 25 ° C.
  • Components of PCR reaction solution [Mix for PCR] The same PCR mix 1 used in Test Example 6 was used.
  • CDNA prepared from HeLa cell (derived from human cervical cancer) RNA was used.
  • Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep TM II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) were used for RNA extraction and cDNA synthesis. I followed the book.
  • reaction solution Each primer / probe was mixed at a ratio of 1/20 and a nucleic acid template at a ratio of 1/20 to Mix 1 for PCR to prepare 19 ⁇ L of a mixed solution.
  • 1 ⁇ L of 20 mM Tris-HCl (pH 7.5) was added to the mixture and exposed at ⁇ 20 ° C. or 25 ° C. for 24 hours.
  • 1 ⁇ L of each 0.1 mg / mL solution was added to the mixed solution (carry-on amount: 0.1 ⁇ g), and the mixture was exposed at 25 ° C. for 24 hours.
  • Test Example 9 Suppression of probe degradation by a small amount of anti-domain E antibody It was confirmed whether or not probe degradation was suppressed when a PCR reaction solution containing 0.1 ⁇ g of anti-domain E antibody was exposed at 25 ° C. for 24 hours.
  • CDNA prepared from HeLa cell (derived from human cervical cancer) RNA was used.
  • Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep TM II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) were used for RNA extraction and cDNA synthesis. I followed the book.
  • Test Example 10 Expression of chimeric anti-domain E antibody (1) Preparation of antibody expression plasmid An antibody sequence containing the CDR of Anti-TH4 was designed, and oligo DNA was obtained by artificial synthesis. Antibodies with heavy and light chain constant regions from mice set forth in SEQ ID NOs: 53 and 54, using the plasmid PowerExpress System TM (MPH-102 and MPL-202, Toyobo Co., Ltd.) according to the accompanying instruction manual. An expression plasmid was prepared.
  • Test Example 11 Suppression of probe degradation by chimeric anti-domain E antibody
  • the PCR reaction solution containing the chimeric anti-domain E antibody was exposed at 25 ° C. for 24 hours, it was confirmed whether the chimeric anti-domain E antibody suppressed the degradation of the probe.
  • reaction solution [mix for PCR] The same PCR mix 1 used in Test Example 6 and PCR mix 2 used in Test Example 7 were used.
  • CDNA prepared from HeLa cell (derived from human cervical cancer) RNA was used.
  • Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep TM II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) were used for RNA extraction and cDNA synthesis. I followed the book.
  • chimeric anti-domain E antibody 1 ⁇ L of a 0.1 mg / mL solution was added to the mixed solution (carry-on amount: 0.1 ⁇ g), and the mixture was exposed at 25 ° C. for 24 hours. Then, using a real-time PCR device (Applied Biosystems 7500 Fast real-time PCR system), the reaction was carried out in the following temperature cycle. The fluorescence value was read in an extension step at 60 ° C. for 60 seconds. (Temperature cycle) Step 1: 95 ° C 1 minute Step 2: 95 ° C 15 seconds-60 ° C 60 seconds 50 cycles (PCR)
  • the RPS19 gene could not be detected in the reaction solutions 4 and 5 to which Tris-HCl was added when exposed at 25 ° C. for 24 hours, but the reaction solutions 4 and 5 to which the chimeric anti-domain E antibody was added were 25 ° C.
  • the RPS19 gene in HeLa cDNA 100, 10, 1, and 0.1 ng could be detected with the same Ct value as when exposed at ⁇ 20 ° C. for 24 hours. Further, when the probe decomposition rate was estimated by the same method as in Test Example 7 (c), it was calculated to be 4.4% for Anti-TAQ2 and 3.3% for Anti-TH4.
  • Test Example 12 Suppression of probe degradation when Taq polymerase (variant) or Z05 polymerase coexists with anti-domain E antibody Exposure to PCR reaction solution containing anti-domain E antibody and Taq polymerase (variant) or Z05 polymerase at 25 ° C for 24 hours. If so, it was confirmed whether the anti-domain E antibody suppressed the degradation of the probe.
  • PCR Mix 4 QuantiNova Probe RT-PCR Kit (QIAGEN, 208352) containing Taq polymerase (mutant).
  • PCR Mix 5 TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, 44445556), including Taq polymerase (mutant).
  • PCR Mix 6 Z05 polymerase (0.05 U / ⁇ L, Roche Diagnostics, HowkZ05, SEQ ID NO: 55); Anti-polymerase antibody for hot start PCR (0.01 ⁇ g / ⁇ L, TCP-101, manufactured by Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.3); 80 mM KCl; 1.5 mM MgCl 2 ; 0.5 mg / mL BSA; 0.1% (v / v) TritonX-100; 0.1% (w / v) sodium cholic acid; and 0.3 mM dNTPs.
  • Z05 polymerase 0.05 U / ⁇ L, Roche Diagnostics, HowkZ05, SEQ ID NO: 55
  • Anti-polymerase antibody for hot start PCR (0.01 ⁇ g / ⁇ L, TCP-101, manufactured by Toyobo Co., Ltd.
  • 10 mM Tris-HCl pH 8.3
  • CDNA prepared from HeLa cell (derived from human cervical cancer) RNA was used.
  • Human HeLa Cell Total RNA (636543, Takara Bio Inc.) and SuperPrep TM II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) were used for RNA extraction and cDNA synthesis. I followed the book.
  • Test Example 13 Inhibition ability of anti-domain E antibody against 5' ⁇ 3'exonuclease activity The obtained anti-domain E antibody was confirmed to have an inhibitory ability of Taq polymerase or Tth polymerase against 5' ⁇ 3'exonuclease activity.
  • reaction solution 9 A Taq enzyme solution containing 1 unit of Taq polymerase (TAP-201, Toyobo Co., Ltd.) and 0.2 ⁇ g of a commercially available hot start antibody Anti-Taq high (TCP-101, Toyobo Co., Ltd.) was prepared. A reaction solution (final concentration: 10 mM Tris-HCl (pH 8. 6), 50 mM KCl, 1.5 mM MgCl 2 ), and the liquid volume was adjusted to 20 ⁇ L (Sample 3). As controls, sample 1 containing neither Taq polymerase enzyme solution nor Anti-TAQ2 and sample 2 containing only Taq polymerase enzyme solution were prepared, and each sample was incubated at 37 ° C. for 24 hours. Then, 100 ⁇ L of 10% (w / v) TCA was added to each sample to precipitate the substrate DNA, and the radioactivity of the free 32P-labeled base remaining in the supernatant was measured.
  • TCA 10%
  • reaction solution 10 A Tth enzyme solution containing 1 unit of Tth polymerase (TTH-301, Toyobo Co., Ltd.) and 0.6 ⁇ g of a commercially available hot start antibody Anti-Taq high (TCP-101, Toyobo Co., Ltd.) was prepared. A reaction solution (final concentration: 10 mM Tris-HCl (pH 8. 6), 50 mM KCl, 1.5 mM MgCl 2 ), and the liquid volume was adjusted to 20 ⁇ L (Sample 3). As controls, Sample 1 containing neither Tth polymerase enzyme solution nor Anti-TH4 and Sample 2 containing only Tth polymerase enzyme solution were prepared, and each sample was incubated at 37 ° C. for 24 hours. Then, 100 ⁇ L of 10% (w / v) TCA was added to each sample to precipitate the substrate DNA, and the radioactivity of the free 32P-labeled base remaining in the supernatant was measured.
  • [Substrate DNA] A reaction solution prepared by mixing 10 ⁇ g of ⁇ DNA and 30 units of ScaI (manufactured by Toyobo Co., Ltd.) according to the instruction manual was prepared and incubated at 37 ° C. for 24 hours. The pellets produced by the phenol / chloroform / isoamyl alcohol (liquid volume ratio 25:24: 1) treatment and ethanol precipitation were dissolved in 100 ⁇ L of TE buffer.
  • Test Example 14 Inhibition ability against 5' ⁇ 3'exonuclease activity when the amount of anti -domain E antibody added was changed. By changing the amount of anti-domain E antibody added, the amount of Taq polymerase 5' ⁇ 3'against exonuclease activity The inhibitory ability was confirmed.
  • reaction solution A Taq polymerase enzyme solution containing 1 unit of Taq polymerase (TAP-201, Toyobo Co., Ltd.) and 0.2 ⁇ g of a commercially available hot start antibody Anti-Taq high (TCP-101, Toyobo Co., Ltd.) was prepared. A mixture of Taq polymerase enzyme solution and Anti-TAQ2 0.05, 0.1, 0.2, or 0.4 ⁇ g containing substrate DNA (9000 cpm, count rate 80%) whose 5'end was radioactively labeled with 32P.
  • sample 1 containing neither Taq polymerase enzyme solution nor Anti-TAQ2 and sample 2 containing only Taq polymerase enzyme solution were prepared, and each sample was incubated at 37 ° C. for 24 hours. Then, 100 ⁇ L of 10% (w / v) TCA was added to each sample to precipitate the substrate DNA, and the radioactivity of the free 32P-labeled base remaining in the supernatant was measured.
  • Results 100% is used for sample 1 containing neither Taq polymerase enzyme solution nor Anti-TAQ2 because the substrate DNA is not degraded, and 0% is used for sample 2 containing only Taq polymerase enzyme solution because the substrate DNA is decomposed most.
  • Table 22 shows the results of calculating the residual rate of the substrate DNA of Samples 3 to 6 as the ability to inhibit 5' ⁇ 3'exonuclease activity.
  • Test Example 15 Suppression of probe degradation of Tth polymerase by an antibody that specifically binds to domain E of Taq polymerase When a PCR reaction solution containing an antibody that specifically binds to domain E of Taq polymerase and Tth polymerase is exposed at 25 ° C. for 24 hours. , It was confirmed whether or not the antibody suppresses probe degradation of Tth polymerase.
  • CDNA prepared from HeLa cell (derived from human cervical cancer) RNA was used.
  • Human HeLa Cell Total RNA (product code: 636543, Takara Bio Inc.) and SuperPrep TM II Cell Lysis & RT Kit for qPCR (SCQ-401, Toyobo Co., Ltd.) were used for RNA extraction and cDNA synthesis.
  • SCQ-401 SuperPrep TM II Cell Lysis & RT Kit for qPCR
  • Test Example 16 Inhibition ability of anti-domain E antibody against 5' ⁇ 3'exonuclease activity when the substrate DNA is changed
  • the double-stranded substrate DNA derived from ⁇ DNA described in SEQ ID NOs: 56 and 57 is designed, and 5 of Taq polymerase or Tth polymerase. The ability of the anti-domain E antibody to inhibit' ⁇ 3'exonuclease activity was confirmed.
  • Activity measurement mix 1 Taq polymerase (0.05U / ⁇ L, TAP-201, manufactured by Toyobo Co., Ltd.); Anti-polymerase antibody for hot start PCR (0.01 ⁇ g / ⁇ L, TCP-101, manufactured by Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.6); 50 mM KCl; 1.5 mM MgCl 2 ; 0.3 ⁇ M double-stranded substrate DNA;
  • Activity measurement mix 2 Tth polymerase (0.05U / ⁇ L, TTH-301, manufactured by Toyobo Co., Ltd.); Anti-polymerase antibody for hot start PCR (0.01 ⁇ g / ⁇ L, TCP-101, manufactured by Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.6); 50 mM KCl; 1.5 mM MgCl
  • a ⁇ DNA-derived double-stranded substrate DNA having the oligonucleotides set forth in SEQ ID NOs: 56 and 57 was designed.
  • the oligonucleotides set forth in SEQ ID NOs: 56 and 57 were synthesized separately and used in equal amounts.
  • S 21 Band intensity after 24 hours exposure at -20 ° C without anti-domain E antibody (corresponding to 24 hours before exposure at 25 ° C)
  • S 22 24 at 25 ° C without anti-domain E antibody Band intensity after time exposure
  • S 23 Band intensity after exposure for 24 hours at 25 ° C with anti-domain E antibody
  • Anti-TAQ2 was added and exposed at 25 ° C for 24 hours with Taq polymerase and Tth polymerase.
  • Test Example 17 Inhibition of DNA polymerase fluorescently labeled double-stranded substrate DNA (probe) degradation by an antibody that specifically binds to domain E of Taq polymerase
  • An antibody that specifically binds to domain E of Taq polymerase and a DNA polymerase (Taq polymerase or Tth) When the PCR reaction solution containing (polymerase) was exposed at 37 ° C. for 24 hours, it was confirmed whether or not the antibody suppressed the degradation of the fluorescently labeled double-stranded substrate DNA of the polymerase.
  • PCR Mix 7 Taq polymerase (0.05U / ⁇ L, TAP-201, manufactured by Toyobo Co., Ltd.); Anti-polymerase antibody for hot start PCR (0.01 ⁇ g / ⁇ L, TCP-101, manufactured by Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.6); 50 mM KCl; 1.5 mM MgCl 2 ; and 0.3 ⁇ M fluorescently labeled double-stranded substrate DNA.
  • Taq polymerase 0.05U / ⁇ L, TAP-201, manufactured by Toyobo Co., Ltd.
  • Anti-polymerase antibody for hot start PCR (0.01 ⁇ g / ⁇ L, TCP-101, manufactured by Toyobo Co., Ltd.
  • 10 mM Tris-HCl pH 8.6
  • 50 mM KCl 1.5 mM MgCl 2
  • PCR Mix 8 Tth polymerase (0.05U / ⁇ L, TTH-301, manufactured by Toyobo Co., Ltd.); Anti-polymerase antibody for hot start PCR (0.01 ⁇ g / ⁇ L, TCP-101, manufactured by Toyobo Co., Ltd.); 10 mM Tris-HCl (pH 8.6); 50 mM KCl; 1.5 mM MgCl 2 ; and 0.3 ⁇ M fluorescently labeled double-stranded substrate DNA.
  • [Fluorescently labeled double-stranded substrate DNA] A fluorescently labeled double-stranded substrate DNA derived from ⁇ DNA having the oligonucleotides of SEQ ID NOs: 58 and 59 was designed (where the 5'end of SEQ ID NO: 58 was labeled with FAM and the 5'end was labeled with BHQ1). .. The oligonucleotides set forth in SEQ ID NOs: 58 and 59 were synthesized separately and used in equal amounts.
  • both Taq polymerase and Tth polymerase were calculated as (e) fluorescently labeled double-stranded substrate DNA degradation rate (%) ⁇ 10%. Therefore, it was confirmed that Anti-TAQ2 exhibits sufficient inhibitory ability to degrade fluorescently labeled double-stranded substrate DNA against both Taq polymerase and Tth polymerase.
  • Test Example 18 Measurement of antibody binding rate constant ka value, dissociation rate constant cd value, and equilibrium dissociation constant KD value Surface plasmon resonance (SPR) was used to determine the affinity of the antibody for Tth polymerase.
  • SPR Surface plasmon resonance
  • a Biacore X100 device (Cytiva) was used.
  • As the running buffer 0.01M HEPES, 0.15M NaCl, 3 mM EDTA, 0.05% (v / v) Surfactant P 20 (Cytiva) was used.
  • Test Example 19 Epitope Mapping of Antibodies Epitope mapping was performed using the conformation epitope mapping of the Peptide Microarray Contract Analysis Service of PEPperMAP TM of PEPperPINT.
  • the amino acid sequence of Taq exo shown in SEQ ID NO: 1 (from the N-terminal of who Taq to the 290th amino acid) and the amino acid sequence of Tth exo shown in SEQ ID NO: 2 (from the N-terminal of whole Tth to the 292nd amino acid).
  • peptides consisting of 7, 10 and 13 amino acids were synthesized on a peptide array by shifting them by 1 amino acid so as to overlap with 6, 9 and 12 amino acids. Then, for each peptide array, the detection signal indicating the binding property of Anti-TAQ2 and Anti-TH4 was measured, and the epitope that interacts with the antibody was identified.
  • Anti-TAQ2 binds to at least two regions of the amino acid sequences KEDGDAVIVVF (SEQ ID NO: 61) and LERREFGSLLHEF (SEQ ID NO: 77) in Taq exo (SEQ ID NO: 1) and also in the amino acid sequence EDGYKAVFVVF in Tth exo (SEQ ID NO: 2). It was confirmed that it binds to at least four regions of (SEQ ID NO: 62), HLITPEWLW (SEQ ID NO: 66), KYGLRPEQWVDF (SEQ ID NO: 67) and LRAFULLERLEF (SEQ ID NO: 78).
  • Anti-TH4 binds to at least three regions of the amino acid sequences HEAYGGY (SEQ ID NO: 64), EKYGLRPDQWADY (SEQ ID NO: 68) and RAFLEXERLEFGSLHL (SEQ ID NO: 80) in Taq exo (SEQ ID NO: 1) and also Tth exo (SEQ ID NO: 80). It can bind to at least five regions of the amino acid sequences HEAYYEY (SEQ ID NO: 65), GLRPEQWVDF (SEQ ID NO: 70), ITPEWLW (SEQ ID NO: 71), LRAFLEXLEF (SEQ ID NO: 78) and LEFGSLHLHEF (SEQ ID NO: 82) in No. 2). confirmed.
  • Anti-TAQ2 contains an epitope containing a sequence in amino acid region A common to or similar to Taq exo (EDGDAVIVVF (SEQ ID NO: 60) or EDGYKAVFVVF (SEQ ID NO: 62)) and a common sequence in amino acid region D (LERLEF (SEQ ID NO: 62)). It was an antibody that recognizes and binds to an epitope containing No. 75)).
  • Anti-TH4 contains an epitope containing a sequence in amino acid region B that is common or similar to Taq exo and Tth exo (HEAYGGY (SEQ ID NO: 64) or HEAYEAY (SEQ ID NO: 65)) and a common sequence in amino acid region C (EKYGLRPDQWADY (SEQ ID NO: 65)).
  • Anti-TAQ2 and Anti-TH4 in the amino acid sequence of Taq exo, it binds to an epitope containing a common sequence in amino acid region D (LERLEFGSLLLH (SEQ ID NO: 76)), and in the amino acid sequence of Tth exo, it binds to an epitope.

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Abstract

L'invention concerne un anticorps capable d'inhiber l'activité exonucléase 5'→ 3' d'une ADN polymérase, ou un fragment de l'anticorps.　L'invention concerne un anticorps capable de se lier spécifiquement à un domaine d'activité exonucléase 5'→ 3' d'une ADN polymérase, ou un fragment de l'anticorps.
PCT/JP2021/045700 2020-12-11 2021-12-10 Anticorps capable de se lier spécifiquement à un domaine actif d'exonucléase 5'→ 3' de l'adn polymérase WO2022124418A1 (fr)

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CN202180082185.3A CN116583600A (zh) 2020-12-11 2021-12-10 与dna聚合酶的5’→3’外切核酸酶活性结构域特异性结合的抗体
EP21903514.4A EP4261226A1 (fr) 2020-12-11 2021-12-10 Anticorps capable de se lier spécifiquement à un domaine actif d'exonucléase 5'? 3' de l'adn polymérase
US17/996,601 US11970549B2 (en) 2020-12-11 2021-12-10 Antibody capable of binding specifically to 5′ to 3′ exonuclease active domain of DNA polymerase

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CN116284413B (zh) * 2022-11-25 2023-09-05 厦门康基生物科技有限公司 一种Taq DNA聚合酶的单克隆抗体F12H12及其应用

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CN115594768A (zh) * 2022-12-12 2023-01-13 珠海宝锐生物科技有限公司（Cn） 一种分泌抗dna聚合酶单克隆抗体的杂交瘤细胞、单克隆抗体及其应用
CN115594768B (zh) * 2022-12-12 2023-03-17 珠海宝锐生物科技有限公司 一种分泌抗dna聚合酶单克隆抗体的杂交瘤细胞、单克隆抗体及其应用

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